Summary Telomerase is an RNA-protein complex that replicates the very ends (telomeres) of the linear chromosomes of humans and other eukaryotes. Telomere maintenance is required for continuous proliferation of cells, and in most human cancers this is achieved by reactivating the gene for the catalytic subunit of telomerase (telomerase reverse transcriptase or TERT). The trigger for reactivation remained mysterious until 2013, when two research groups reported mutations in the promoter of the TERT gene present in multiple tumor types but not in adjacent healthy tissue. Recently we reported that these TERT promoter mutations were indeed associated with higher production of TERT protein, higher telomerase enzyme activity, and longer telomeres in urothelial cancer (UC), and associated with poor patient survival. However, very little is known about the molecular mechanisms by which the promoter mutations lead to TERT activation, which has impeded progress both in basic science and in clinical applications. Now we propose to unravel the mechanisms of TERT activation in UC and hepatocellular carcinoma (HCC) cell lines. Many of these cell lines are heterozygous for the TERT promoter mutation ? i.e., only one of the two TERT genes in the cell has the mutation. Thus, we will compare the two alleles to test for differences in RNA polymerase II occupancy, marks of epigenetic silencing, and production of mRNA, allowing a specific hypothesis about TERT gene silencing and reactivation to be tested. To move beyond association and test for causation, we will use CRISPR-Cas9 genome editing to insert or erase TERT promoter mutations and assess whether this is sufficient to switch TERT production. If the promoter mutation is not sufficient, we will inactivate factors such as the histone methyltransferase PRC2 (which adds repressive chromatin marks) either by knockdown or by adding a validated small-molecule inhibitor and test for reactivation. Furthermore, bioinformatics (gene expression data) reveals candidates for other components involved in gene reactivation that will be tested. The long-term goal of this project is to understand the state of the repressed and activated TERT genes and the steps involved in telomerase reactivation in cancer. This basic science study will provide information about cancer biology and is likely to give information that is clinically relevant.